Molecular assays for the detection of prostate tumor derived nucleic acids in peripheral blood

The C4-2 prostate cancer cell line [25] was a gift from Leland Chung, Ph.D. (Emory University). LNCaP cells [26] were obtained from the American Type Culture Collection (ATCC; Manassas, VA). GFP-LNCaP cells were kindly provided by Dr. Srivastava, Center for Prostate Disease Research (Rockville, MD). All cell lines were grown under standard culture conditions in RPMI 1640 medium supplemented with 10% fetal bovine serum (ATCC), and with G418 (0.5 mg/mL; Invitrogen, Carlsbad, CA) for GFP-LNCaP cells.

GFP-LNCaP cells were treated by mild trypsinization (Trypsin/EDTA, Invitrogen, Carlsbad, CA) from one 25 mm² dish (BD, Franklin Lakes, NJ) and harvested by sedimentation (500 g, 5 min, room temperature). The cell pellet was resuspended in PBS (dPBS, Invitrogen, Carlsbad, CA). C4-2 cells were harvested as described above for GFP-LNCaP cells. Individual cells (n = 5, 10, 25) were aspirated with a micromanipulator pipette system (TransferMan NK, Eppendorf and Leica DMIRB inverted microscope) and added to normal donor blood treated with EDTA to prevent coagulation. Freshly harvested LNCaP cells were transferred to a 10 cm Petri dish containing RPMI 1640 medium at room-temperature. Individual cells were aspirated manually with a standard laboratory pipette under low microscopic magnification (Olympus CK2 equipped with 10× lens) and added to EDTA-treated normal donor blood. Immunomagnetic cell isolation was carried out as described in more detail below. Briefly, for each of the cell lines tested individually in the model system, freshly resuspended cultured cells were added to 1 mL EDTA blood (approximately 100 cells) and incubated with 10 uL anti-EpCAM coated magnetic particles (Invitrogen, Carlsbad, CA) for 20 min at room temperature. Magnetic-bound fractions were washed three times, as described below, and then the washed particles were subjected to fluorescence microscopy as described below.

Specimen processing included CTC enrichment and cell lysis. For specimens that contained C4-2 cells, specimen processing was performed at the University of Washington (Seattle, WA) and the processed specimens were shipped to Gen-Probe Incorporated (San Diego, CA) on dry-ice for further testing. Processing of specimens that contained LNCaP cells or GFP-LNCaP cells was done at Gen-Probe Incorporated.

For visualization experiments of magnetically enriched cells, about 20 μL Mowiol mounting medium [27] was added to the washed magnetic particles. The resulting suspension was spotted onto a glass slide and covered with a round glass cover slip (18 mm diameter) to prevent drying. The mounted cells were visualized with a fluorescence microscope with 10× and 40× lenses (Axio Imager Z1, Zeiss, Thornwood, NY). Pictures were taken with an attached CCD camera (Metasystems, Waltham, MA). GFP-LNCaP cells in EDTA-treated blood were visualized after spreading a thin film onto a glass slide without a cover slip to prevent cellular rupture.

Specimen collection was carried out at the University of Washington under IRB approved protocols that included written informed consent from each of the subjects. This study included consecutive patients who consented and fell within the inclusion and exclusion guidelines. In total, sixty-five men diagnosed with prostate cancer participated in this study: thirty-five men who had been diagnosed with advanced-stage prostate cancer by bone scan (hereafter referred to as advanced PCa) and twenty-nine who had been diagnosed as early-stage cancer patients by biopsy following a serum PSA determination and digital rectal exam (hereafter termed pre-radical retropubic prostatectomy (pre-RP)). Moreover, five men diagnosed with benign prostatic hyperplasia (BPH) were included in the study. Five age-matched healthy individuals served as controls for specificity of cell capture and molecular analysis. Blood from apparently healthy volunteers was used as a matrix for experiments in the model system (described above) that tested samples containing known amounts of cultured C4-2, LNCaP, and GFP-LNCaP cells.

Whole blood was collected by venipuncture into two 10 ml EDTA-containing Vacutainer^® tubes (BD, Franklin Lakes, NJ) and stored on ice up to 4 hours before processing. The collected blood was pooled and then processed by immunocapture and plasma generation as described in detail below. In cases when only one vacutainer tube could be obtained, the processing procedure was: (1) perform immunomagnetic cell capture on 5 mL blood with anti-EpCAM and anti-PSMA coated particles, (2) plasma preparation from up to 4 mL blood, and (3) perform immunomagnetic CTC isolation with anti-EpCAM coated particles by using the remaining whole blood if sufficient volume remained.

Anti-EpCAM coated magnetic particles were purchased from Invitrogen (Carlsbad, CA). Anti-PSMA coated particles were generated by reacting a monoclonal anti-PSMA antibody (Beckman Coulter) with human anti-mouse magnetic particles according to manufacturer's specifications (Invitrogen, Carlsbad, CA).

For CTC isolation, 5 mL or 7.5 mL of whole blood was incubated with 100 uL antibody-coated magnetic particles (anti-PSMA and/or anti-EpCAM) for 30 min at room temperature. Magnetic-bound fractions were subjected to three washing steps with phosphate buffered saline (dPBS; Invitrogen) containing 0.2% (w/v) BSA (Jackson Immuno Research, West Grove, PA) and subsequently treated with Gen-Probe lysis buffer. The immunomagnetically-enriched fractions were stored at - 20°C until mRNA isolation was performed (see below).

Blood specimens (up to 4 mL of EDTA-treated blood) were subjected to sedimentation at 1,600 g for 10 min at 4°C to generate plasma. The plasma fraction was carefully separated from the cellular fraction by standard laboratory pipetting with a 1 mL tip and mixed with an equal volume of Gen-Probe lysis buffer. The treated plasma was stored at -20°C until mRNA isolation was carried out (see below).

TMPRSS2 (T2):ERG gene fusion, PCA3 and PSA mRNAs were either detected qualitatively or quantitatively using assays that included the steps of magnetic target capture, transcription-mediated amplification (TMA), and a hybridization protection assay. Specifically, target mRNA was purified from immunomagnetically enriched and processed plasma fractions by hybridization to magnetic particles via target-specific oligonucleotides (target capture step), amplified by TMA, and detected by using target-specific acridinium ester (AE)-labeled probes (hybridization protection assay step) as described in [18]. Three T2:ERG splice variants were detected qualitatively, T2:ERGa, b and c [28], also known as Types III, I and VI, respectively [29]. Amplification primers for T2:ERG gene fusion mRNA were located in T2 exon 1 and ERG exon 4 (T2:ERGa), T2 exon 1 and ERG exon 2 (T2:ERGb), and T2 exons 1 and 2 and ERG exon 4 (T2:ERGc). The AE-labeled probes for each T2:ERG splice variant spanned the junction between T2 and ERG. Primers for PCA3 mRNA targeted exons 3 and 4, with the AE-labeled probe spanning the exon 3/4 junction. Primers for PSA mRNA targeted exons 2 and 3, with the AE-labeled probe spanning the exon 2/3 junction. PCA3 and PSA mRNAs were detected quantitatively (signal-to-noise set to 2-fold or greater), whereas T2:ERG was a qualitative assay (cutoff = 100,000 relative light units, RLUs). Calibrators and controls consisted of T2:ERG, PCA3 or PSA in vitro transcripts (IVTs) in detergent solution. The T2:ERGa, b and c IVTs were prepared from plasmids provided by A. Chinnaiyan (University of Michigan) [28]. IVT copy levels were calculated based on spectroscopic concentration determination using A₂₆₀ measurements. Assays were performed at Gen-Probe Incorporated using its DTS^® 400 Systems and assay protocol for reagent addition volumes and incubation times and temperatures as previously described in [18].

We developed an immunomagnetic particle capture system for the isolation of rare cells out of blood based on antibody coated magnetic particles directed to epithelial and prostate cell surface epitopes. To assess successful isolation of cultured prostate cancer cells from a mixture of normal blood cells, we used microscopic or molecular methods.

GFP-expressing LNCaP cells were added to normal donor blood and subsequently visualized before and after immunomagnetic enrichment (Figure 1A, and Figure 1B, C, respectively). As expected, the magnetic particle-bound fraction contained fluorescent GFP-LNCaP cells (Figure 1B) and only a small amount of non-fluorescent structures, possibly representing non-specifically bound blood cells (Figure 1C).

We next demonstrated molecular detection of prostate cancer cells with Gen-Probe's protocol that includes target capture, TMA and hybridization protection assay steps. PSA mRNA was detected in five C4-2 cells that had been immunomagnetically enriched from 5 mL of normal donor blood. The measured PSA mRNA copy numbers correlated well with cellular input (Figure 1D).

To investigate the specificity of immunomagnetic capture, we mixed LNCaP cells with normal donor blood and subjected the mixture to the enrichment steps using control magnetic particles that were devoid of anti-EpCAM or anti-PSMA primary antibodies. No PSA mRNA was detected in this condition, indicating that cell capture was antibody dependent (Figure 1E). Moreover, PSA mRNA was undetectable in magnetically enriched fractions from normal blood donors that were devoid of added cultured prostate cancer cells (Figure 1E). Both anti-EpCAM and anti-PSMA coated magnetic particles gave equivalent performance in these model system experiments (data not shown).

We next investigated the CTC enrichment method with freshly drawn blood specimens from prostate cancer patients. Samples were processed on site within four hours from time of collection. For those specimens with sufficient blood volume, we processed the specimens in three fractions: (1) CTC enrichment using anti-EpCAM plus anti-PSMA magnetic particles on whole blood; (2) CTC enrichment using anti-EpCAM magnetic particles alone on whole blood; and (3) plasma fraction using standard centrifugation. For specimens with insufficient blood volume, we omitted the second fraction (anti-EpCAM magnetic particles alone). Processed fractions were then tested using amplified molecular assays.

Results for the PSA mRNA assay are summarized in Figure 2. As shown in Figure 2A, the combined anti-EpCAM plus anti-PSMA magnetic particle formulation detected 9/16 androgen-independent (56%) and 3/17 androgen-dependent samples (18%). BPH and early stage prostate cancer specimens were devoid of detectable PSA mRNA (0/5 and 0/29, respectively). Results from matched plasma fractions are shown in Figure 2B. In this case, we detected 8/16 androgen-independent (50%) and 2/17 androgen-dependent specimens (12%). Positive plasma fractions were in almost complete concordance with CTC enriched fractions, although CTC enrichment generally gave higher PSA mRNA copy numbers.

Figure 3 shows a comparison of PSA mRNA signals from CTC enriched samples where sufficient blood volume had been collected from the patient to allow a comparison between the single antibody (anti-EpCAM) and dual antibody (anti-EpCAM plus anti-PSMA) magnetic particle formulations. The single antibody formulation detected 8/14 cases (57%); whereas the dual antibody formulation detected 7/14 cases (50%), with the majority of positives from androgen-independent patients. We were unable to measure any synergistic effect of the dual antibody magnetic bead formulation in this experiment (see Discussion).

A comparison of PSA, PCA3 and TMPRSS2:ERG mRNA signals is shown in Tables 1 and 2 for blood samples from men with advanced prostate cancer that were processed using the dual antibody magnetic particle formulation. Results from men with androgen-dependent prostate cancer are summarized in Table 1. These men all tested negative for PCA3, whereas one of the men was positive for TMPRSS2:ERG gene fusion mRNA. In contrast, more of the men with androgen-independent prostate cancer tested positive for all three markers (Table 2). PCA3 was positive in 5/16 (31%) of these patients, although PCA3 mRNA copy numbers were at least one order of magnitude lower than PSA mRNA copy numbers in these patients. Three of these patients tested positive for TMPRSS2:ERG gene fusion mRNA in CTC enriched samples (Table 2) and also in plasma (data not shown). When any of the three markers were positive the detection rate increased to 63% (10/16 positive; Table 2). This demonstrates feasibility of applying a research prototype TMA-based molecular assay to CTC enriched patient samples.